1. Field of the Invention
The present invention relates to methods of inhibiting viral entry and spread in vivo and in vitro. The invention further relates to isolated peptides from a mammalian Rho protein which have been found by the inventors to be useful for inhibiting entry of enveloped viruses, specifically paramyxoviruses and lentiviruses, into susceptible cells. In particular embodiments, the invention also relates to methods of preventing infection by enveloped viruses. These methods utilize the inhibitory effect of specific RhoA peptides or specific peptides isolated from the fusion glycoproteins of respiratory syncytial virus (RSV) or human immunodeficiency virus (HIV) on the viral entry mechanism of enveloped viruses.
2. Description of the Related Art
Paramyxoviruses and lentiviruses are important agents of clinical and veterinary disease. These viruses include important human pathogens such as respiratory syncytial virus (RSV), parainfluenza viruses, measles, mumps, HIV-1 and HIV-2, and veterinary pathogens such as bovine RSV, turkey rhinotracheitis virus, Newcastle""s disease virus, rinderpest virus, canine distemper virus, the new morbilliviruses described in seals and horses, and simian immunodeficiency virus (SIV).
The major cause of serious lower respiratory tract illness in infants and immunosuppressed individuals is a paramyxovirus known as respiratory syncytial virus (RSV). Worldwide, RSV causes 65 million infections and 1 million deaths annually. The greatest incidence of disease from RSV infection is from 6 weeks to 6 months of age, with approximately 90,000 children hospitalized each year in the United States with infections caused by RSV. 4500 of those children die. Exaggerated RSV IgE response during RSV bronchiolitis in infancy has also been associated with the widespread problem of recurrent wheezing in early childhood.
Reinfections with RSV are more frequent than with most other viruses of the respiratory tract. Serious disease is usually associated with the first or second infection. Although disease severity declines with repeated infection, previous infection with RSV does not prevent illness in subsequent infections. Immunity is apparently incomplete. Live virus vaccines have generally proven to be inadequately immunogenic by the time they have been attenuated to a sufficient level to produce no clinical illness. A formalin-inactivated vaccine developed in the 1960s not only failed to produce a protective response against the virus, but induced exacerbated disease in vaccinated children during a subsequent epidemic, and some attenuated RSV strains have the potential to revert to virulence after human passage. Vaccine development has therefore been approached cautiously, although efforts to prevent RSV disease in infants and young children have continued to target active immunization with an inactivated vaccine, a live attenuated virus vaccine, or a subunit vaccine, and passive immunization of the fetus by active immunization of the mother with a human monoclonal RSV antibody or hyperimmune RSV immune globulin.
High-risk infants are treated with immunoglobulin (IG) to protect against RSV, but intravenous RSV IG is very expensive and administration requires a monthly infusion lasting 7 hours or more to maintain acceptable antibody titers.
While RSV poses a serious health threat, a more deadly infection is established by the lentiviruses known as human immunodeficiency viruses HIV-1 and HIV-2, which cause acquired immunodeficiency syndrome (AIDS). The World Health Organization estimates that 16,000 new HIV-1 infections in humans occur daily. Although some individuals have been identified in whom the infection has progressed slowly, the fatality rate for infected individuals is considered to be 100 percent.
Vaccine development for the prevention of HIV infection, and subsequent development of acquired immunodeficiency syndrome (AIDS), has been less successful than had earlier been anticipated. The present generation of gp120 subunit vaccines have not been shown to induce relevant antibody or cell-mediated immune responses of significant potency, and their performance in Phase I/II trials has been disappointing. Contributing to the difficulties in vaccine development are the characteristics of the virus, including poor immunogenicity of the HIV envelope glycoproteins and their resistance to neutralizing antibodies, the extensive variation in the viral genome, and the ability of the virus to become integrated into the host genome of immune cells (Agosto et al.).
One of the most promising, although very costly ($10,000 to $12,000 per year), treatments for HIV infection is known as highly active antiretroviral therapy (HAART), which combines the effects of nucleoside reverse transcriptase inhibitors and protease inhibitors. Even with this very aggressive treatment regimen, however, unintegrated HIV-1 DNA in cells from patients receiving HAART treatment has been found, suggesting that a low level of viral replication may continue and contribute to the maintenance of a reservoir of HIV-infected cells (Chun et al.).
Blocking the binding of the virus also has proven to be more difficult than anticipated. Early experiments had shown that HIV""s primary receptor, CD4, was not itself sufficient to promote viral entry into a susceptible cell. Subsequently, coreceptors were identified, making earlier hopes to develop drugs to block viral attachment more difficult to realize. To date, more than a dozen chemokine receptor coreceptors for HIV gp120 have been found (Balter). The type of receptor used may even vary during the course of the viral infection (Owen et al.).
Identification of a specific fusion receptor could lead to the development of a more effective method of inhibiting HIV infection than have previous efforts to inhibit HIV infection by means of an attachment receptor, and inhibition of RSV infection via a specific fusion receptor provides a safer, more effective means of disease prevention than current vaccine and treatment techniques.
The present invention seeks to overcome these and other shortcomings inherent in the prior art by use of isolated peptides which inhibit viral infection of a susceptible cell. In the present invention, isolated peptides are derived from the mammalian RhoA protein sequence. More specifically, these peptides represent amino acid sequences within, and overlapping N-terminal and C-terminal to, the viral fusion protein binding domain of the RhoA protein. The RhoA viral fusion protein binding domain of the present invention is defined generally by amino acids 67-109 of the RhoA protein, with a core binding sequence located at residues 80-89. Furthermore, these peptides include, but are not limited to, a peptide comprising amino acid residues 77-95 of the RhoA protein.
Also provided by the present invention are peptides corresponding to amino acid sequences within, or overlapping N-terminal or C-terminal to, the RhoA binding domain of a viral fusion protein. These peptides may be defined by amino acid sequences from the F glycoprotein of respiratory syncytial virus, the gp41 fusion protein of human immunodeficiency virus, or the fusion proteins of other viruses which utilize a RhoA-mediated mechanism of cellular entry. The peptides include an isolated peptide corresponding to amino acids 35 to 50 of the human immunodeficiency virus glycoprotein, gp41, and an isolated peptide corresponding to amino acids 9 to 18 of the F1 subunit of the F glycoprotein of respiratory syncytial virus.
In the method of the present invention, isolated RhoA peptides and/or isolated viral fusion protein peptides are administered to a subject to inhibit viral infection by enveloped viruses, including respiratory syncytial virus and human immunodeficiency virus. Because these and other viruses, particularly the enveloped viruses, share a common cellular entry mechanismxe2x80x94fusion of the viral envelope and the cell membranexe2x80x94the RhoA peptides inhibit viral entry for other viruses which are demonstrated to share the cellular entry mechanism common to respiratory syncytial virus (RSV) and human immunodeficiency virus (HIV).
Peptides of the present invention are administered in therapeutically effective dosages to a subject at risk for viral infection or a subject in whom active infection has already been established. In the subject at risk for infection, the peptides inhibit viral entry into susceptible cells and subsequent infection. In the subject in whom active infection has been established, the peptides inhibit cell-to-cell spread of virus and subsequent infection of additional cells.
Peptides, antibodies, and mimetic or peptidomimetic/peptoid compounds of the present invention are delivered by various routes, including, but not limited to, intravenously, orally, nasally, parenterally, and topically. More specifically, topical administration may include a liquid preparation for administration to a puncture wound, such as a needle prick, or cut. Topical administration may also include spermicidal or microbicidal jelly, to which the peptide, mimetic or peptidomimetic/peptoid of the present invention has been added, for use during sexual intercourse. Oral administration may include peptide complexed with a pharmaceutically acceptable carrier and delivered in tablet, caplet, or capsule form. More specifically, to protect the tissues of the intestinal mucosa, peptides, mimetics, or peptidomimetics/peptoids may be administered in enteric-coated capsules, caplets, or tablets. Oral administration also may include administration by aerosol spray or mouthwash, when peptide, mimetic or peptidomimetic/peptoid has been combined with a pharmaceutically acceptable carrier. Peptides, mimetics, or peptidomimetics/peptoids of the present invention may also be administered nasally, by nasal drops or nasal spray.
RhoA peptides, or viral fusion protein peptides as described by the present invention, in combination with appropriate immunogenic adjuvants, are administered to a subject at risk for viral infection to produce antibodies which inhibit viral infection. Anti-RhoA peptide antibodies and anti-viral fusion protein peptide antibodies also provide a means of passive immunization for individuals, such as very young children and immunosuppressed individuals, at increased risk of viral infection following exposure. Anti-idiotypic antibodies generated by peptides of the present invention also inhibit infection, as well as providing appropriate probes to determine antibody titers in immunized individuals.
RhoA peptides also may be used to identify the RhoA binding region of viral fusion proteins. Isolated peptides from these viral proteins are used as immunogens in vaccines to produce antibodies to inhibit viral infection in the immunized subject.
RhoA peptides, antibodies, and anti-idiotypic antibodies of the present invention can be used to screen blood and tissue samples for the presence of virus, as well as identify viruses which bind to the RhoA protein and are therefore likely to be susceptible to the inhibitory effects of the Rho or viral fusion peptides, antibodies, mimetics, or peptidomimetics/peptoids. The peptide, and anti-RhoA peptide antibodies, provide a rapid screening method for identifying other viruses for which the peptide will provide inhibition of viral infection in the manner that has been demonstrated for both respiratory syncytial virus and for human immunodeficiency virus. In the case of a subject infected with an unidentified viral agent, the peptide and anti-RhoA antibodies provide a rapid screening method for determining whether the viral agent will be inhibited by treatment with the RhoA peptide, viral fusion peptide, anti-RhoA peptide antibody, anti-viral fusion protein peptide antibody, mimetic, peptidomimetic, or peptoid.
RhoA peptides of the present invention are produced by means not limited to solid-phase synthesis and recombinant DNA methods. Peptidomimetic, peptoid mimetic, and other compounds which imitate the inhibitory efects of RhoA and viral fusion peptides are produced using solid phase and non-solid phase combinatorial methods, and identified using screening methods including, but not limited to high-throughput screening by ELISA, by size exclusion chromatography (when bound to the appropriate RhoA or viral fusion protein target), and by phage display library screening methods.